Mitotic cell division is fundamental to the growth and development of eukaryotic organisms. The final crucial step in cell division that results in cytoplasmic separation of progeny cells is a process known as cytokinesis. Cytokinesis in animal cells and yeast require membrane constriction driven by an actomyosin-based contractile ring and targeted membrane insertion at the site of cell cleavage. Successful cell division depends on spatial and temporal coordination of these cytokinetic events with chromosome segregation. We have been using the budding yeast Saccharomyces cerevisiae as the model to study the intricate structural and regulatory steps required for cytokinesis. We have shown that assembly of the contractile ring in budding yeast occurs in two temporally separate stages during which myosin-II and actin filaments assemble into a ring around the bud neck connecting the mother and daughter cells. Contraction of this ring at the onset of cytokinesis is regulated by the mitotic exit network. The exocytic pathway is also directed to the bud neck around the time of cytokinesis, and preliminary results suggest that this event is required for cytokinesis. Three specific aims are proposed. First, we will further investigate how myosin-II is recruited to the site of cell division at the G1/S transition and how actin filaments are assembled in the contractile ring during anaphase. Second, we will try to identify the molecular target(s) of the mitotic exit network in triggering the onset of cytokinesis. Finally, we will investigate how the exocytic pathway is directed toward cell division site during mitosis. The experimental approach involves a combination of genetic, biochemical, and microscopy imaging techniques. Because the cell cycle regulatory pathways and the structural components of the cell division site are similar between yeast and animal cells, our study is likely to provide valuable insights into the conserved mechanisms that control cytokinesis in eukaryotes.